Ion-Exchange Chromatography: Basic Principles and Application to the Partial Purification of Soluble Mammalian Prolyl Oligopeptidase

Abstract

Ion-exchange chromatography (IEC) allows for the separation of ionizable molecules on the basis of differences in charge properties. Its large sample-handling capacity, broad applicability (particularly to proteins and enzymes), moderate cost, powerful resolving ability, and ease of scale-up and automation have led to it becoming one of the most versatile and widely used of all liquid chromatography (LC) techniques. In this chapter, we review the basic principles of IEC, as well as the broader criteria for selecting IEC conditions. By way of further illustration, we outline protocols necessary to partially purify a serine peptidase from bovine whole brain cytosolic fraction, covering crude tissue extract preparation through to partial purification of the target enzyme using anion-exchange chromatography. Protocols for assaying total protein and enzyme activity in both pre- and post-IEC fractions are also described. The target serine peptidase, prolyl oligopeptidase (POP, EC3.4.21.26), is an 80-kDa enzyme with endopeptidase activity towards peptide substrates of ≤30 amino acids. POP is a ubiquitous post-proline cleaving enzyme with particularly high expression levels in the mammalian brain, where it participates in the metabolism of neuroactive peptides and peptide-like hormones (e.g. thyroliberin, gonadotropin-releasing hormone).

Ion-Exchange Chromatography and Its Applications

“…Ion-exchange chromatography which is designed specifically for the separation of differently charged or ionizable compounds comprises from mobile and stationary phases similar to other forms of column based liquid chromatography techniques [9][10][11]. Mobil phases consist an aqueous buffer system into which the mixture to be resolved.…”

“…Mobil phases consist an aqueous buffer system into which the mixture to be resolved. The stationary phase usually made from inert organic matrix chemically derivative with ionizable functional groups (fixed ions) which carry displaceable oppositely charged ion [11]. Ions which exist in a state of equilibrium between the mobile phase and stationary phases giving rise to two possible formats, anion and cation exchange are referred to as counter ion ( Figure 1) [1,13].…”

“…Ions which exist in a state of equilibrium between the mobile phase and stationary phases giving rise to two possible formats, anion and cation exchange are referred to as counter ion ( Figure 1) [1,13]. Exchangeable matrix counter ions may include protons (H [11]. Cations are separated on cation-exchange resin column and anions on an anion exchange resin column [10].…”

“…During ion exchange chromatography for example in anion exchange as illustrated in Figure 2, negatively charged protein analytes can be competitively displaced by the addition of negatively charged ions. The affinity of interaction between the salt ions and the functional groups will eventually exceed that the interaction exists between the protein charges and the functional groups, resulting in protein displacement and elution by increasing gradually the salt concentration in the mobile phase [11].…”

“…The earliest report of ion-exchange chromatography date back to 1850, Thompson studied the adsorption of ammonium ions to soils [9][10][11]. Spedding and Powell published a series of papers describing practical methods for preparative separation of the rare earths by displacement ionexchange chromatography in 1947.…”